CN112168959B

CN112168959B - Porcine parvovirus and encephalitis B bigeminy subunit vaccine and preparation method and application thereof

Info

Publication number: CN112168959B
Application number: CN202010996193.6A
Authority: CN
Inventors: 周明光; 张华伟; 陈映瑾; 徐高原; 曾小燕; 朱娴静; 金建云; 陈章表
Original assignee: Wuhan Keqian Biological Co ltd
Current assignee: Wuhan Keqian Biological Co ltd
Priority date: 2020-09-21
Filing date: 2020-09-21
Publication date: 2022-05-03
Anticipated expiration: 2040-09-21
Also published as: CN112168959A

Abstract

The invention relates to a bivalent subunit vaccine of porcine parvovirus and Japanese encephalitis as well as a preparation method and application thereof. According to the invention, the nucleotide sequences of the encoded porcine parvovirus VP2 protein and the Japanese encephalitis E protein are optimized, and the insect-baculovirus eukaryotic expression system is used for expression after the signal peptide GP64 is matched, so that the porcine parvovirus VP2 protein and the Japanese encephalitis E protein are completely consistent in structure, function and natural state, the expression quantity is obviously improved, the immunogenicity is retained to the maximum extent, a bivalent subunit vaccine prepared by matching an adjuvant can play a good immune protection role on the porcine parvovirus and the Japanese encephalitis, and the protection effect is better than that of the existing porcine parvovirus inactivated vaccine and the Japanese encephalitis virus attenuated vaccine. Compared with monovalent vaccine, has the advantages of simplicity, economy, practicality and stress reduction.

Description

Porcine parvovirus and encephalitis B bigeminy subunit vaccine and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological products for livestock, in particular to a porcine parvovirus and Japanese encephalitis bivalent subunit vaccine and a preparation method and application thereof.

Background

Porcine Parvovirus (PPV) is a member of the genus parvovirus of the family parvoviridae, a small non-enveloped virus with a single-stranded DNA genome structure with a virion diameter of about 20nm and a genome of about 5.2 Kb. PPV has two Open Reading Frames (ORFs), including a nonstructural protein (NS1) and a major structural protein (VP2), and VP2 is the major target for the induction of neutralizing antibodies, a virus that is the major epidemic disease causing the reproductive disorders of swine. The disease is mainly characterized by infection and death of embryos and fetuses, particularly the birth sow suffers from dead fetus, malformed fetus and mummy fetus, but the sow has no obvious symptoms per se. The virus has strong resistance to external physical and chemical factors and resistance to lipid solvents (such as diethyl ether, chloroform and the like); has certain resistance to acid, formaldehyde vapor and ultraviolet rays; but can be killed in 0.5% bleaching powder or sodium hydroxide solution for 5 min.

Japanese Encephalitis Virus (JEV) belongs to the genus Flaviviridae of the family Enfaviridae, and is spherical, the nucleic acid is single-stranded RNA, the genome has a total length of 11kb, the outer layer is coated with an envelope, the surface of the envelope is coated with hemagglutinin, the diameter of the envelope is 40nm, the envelope is internally provided with a core consisting of capsid protein (C) and the nucleic acid, the outer layer is coated with a lipid-containing envelope, the surface is coated with envelope glycoprotein (E) spike, namely the viral hemagglutinin, the E protein is coated on the surface of the virus, the virulence and the host range of the virus are determined, the virus can stimulate the production of neutralizing antibodies, and the envelope is internally provided with inner membrane protein (M) which is involved in the assembly of the virus. Abortion and stillbirth of pregnant sows occur after infection, orchitis occurs to boars, persistent hyperpyrexia of fattening pigs and encephalitis of newborn piglets can also be caused, Japanese encephalitis virus has weak resistance to heat, and viruses can be inactivated by diethyl ether, 1:1000 sodium deoxycholate and common disinfectants. Is unstable under acidic conditions and is suitable for pH8.5-9.0.

The two viruses are important infectious diseases causing sow reproductive disorders, are spread in swinery, mainly cause dead fetuses, malformed fetuses and mummy fetuses of sows, cause great manager loss to pig farms and seriously restrict the development of the pig industry. At present, the prior art mainly relies on vaccination to prevent and control the infectious diseases, but in recent years, the phenomenon of mixed infection of a plurality of pathogens of pigs is more and more common, so that the disease condition is more and more complicated, not only great difficulty is brought to clinical diagnosis and prevention and control, but also great economic loss is brought to a pig farm. At present, PPV inactivated vaccine and JEV attenuated single vaccine are mainly immunized respectively for preventing and treating the two diseases, so that the immunization times are increased, the stimulation to the pig body is increased and the stress response is generated on the pig body in a large-scale pig farm; in addition, the Japanese encephalitis virus vaccine is a live vaccine, and the possibility of vaccine virulence reversion is enhanced by frequent and multiple use.

Disclosure of Invention

In order to at least solve the problems in the prior art, the invention provides a bivalent subunit vaccine of porcine parvovirus and Japanese encephalitis and a preparation method and application thereof.

In a first aspect, the present invention provides an immunogenic composition comprising: porcine parvovirus VP2 protein and Japanese encephalitis E protein;

the porcine parvovirus VP2 protein and the Japanese encephalitis E protein are prepared by using the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 is obtained by expressing in an insect-baculovirus eukaryotic expression system.

The scheme uses a baculovirus expression system to express PPV VP2 and JEV E protein, and provides a technical basis for preparing the porcine parvovirus VP2 and Japanese encephalitis virus E protein bigeminy genetic engineering vaccine. The Bac-to-Bac baculovirus expression system is characterized in that a foreign gene is inserted into a baculovirus genome in vitro by using Tn7 transposon action, and the operation is convenient, quick and effective, so that the Bac-to-Bac baculovirus expression system is the most widely used expression system at present. Meanwhile, the large-scale suspension culture technology of insect cells is mature and applied, so that the application of a baculovirus expression system is wider. The baculovirus vector expression system uses a P10 promoter or a PH promoter, inserts an exogenous target gene into the downstream of the promoter in a single copy or multi-copy mode, obtains recombinant baculovirus by a method of enzyme digestion and enzyme ligation, and enables the exogenous gene to be efficiently expressed while the recombinant baculovirus is infected in insect cells or insect bodies.

In addition, PPV VP2 and JEV E proteins are optimized according to the preference of baculovirus codons, and then an AcMNPV GP64 signal peptide sequence is further introduced at the amino terminal of the protein, and an 8 His tag peptide sequence is introduced at the carboxyl terminal of the protein, so that the expression levels of the protein and the protein are effectively improved.

Further, the molar ratio of the porcine parvovirus VP2 protein to the Japanese encephalitis E protein is 0.5-1.5: 1-2.

Further, the content of the porcine parvovirus VP2 protein is 20-40 mug/mL, and the content of the Japanese encephalitis E protein is 40-60 mug/mL.

Further, the eukaryotic expression system uses pFastdual vector to construct recombinant shuttle vector, and/or the eukaryotic expression system uses insect cell High Five^TMExpression of the target protein is carried out.

In a second aspect, the invention provides a porcine parvovirus and encephalitis B bivalent subunit vaccine, which comprises the immunogenic composition and an adjuvant.

Further, the adjuvant is an ISA 15VG adjuvant, and the mass ratio of the sum of the mass of the porcine parvovirus VP2 protein and the mass of the Japanese encephalitis E protein to the adjuvant is 3-5: 1.

In a third aspect, the present invention provides a method for preparing the bivalent subunit vaccine of porcine parvovirus and encephalitis B, comprising:

converting SEQ ID NO:1 and SEQ ID NO:2 to a recombinant vector, expressing through an insect-baculovirus eukaryotic expression system to obtain porcine parvovirus VP2 protein and Japanese encephalitis E protein, and preparing a bivalent subunit vaccine by matching with an adjuvant.

Further, the preparation method comprises the following steps:

(1) converting SEQ ID NO:1 and SEQ ID NO:2 to pFastdial carrier to obtain recombinant plasmids pFastdial-PPV VP2 and pFastdial-JEV E;

(2) respectively transfecting recombinant plasmids pFastdeal-PPV VP2 and pFastdeal-JEV E into an escherichia coli competent cell DH10Bac to prepare recombinant bacmid rBac-PPV VP2 and rBac-JEV E;

(3) transfecting recombinant bacmid rBac-PPV VP2 and rBac-JEV E into sf9 insect cells by utilizing liposome transfection hair, culturing at 25-29 ℃ for 48-72h, collecting cell culture supernatant to obtain recombinant baculovirus, and inoculating the recombinant baculovirus into the insect cells High Five^TMThe expression of the target proteins PPV VP2 and JEV E is carried out;

(4) and (4) preparing the target proteins PPV VP2 and JEV E expressed in the step (3) into a bivalent subunit vaccine by matching with an adjuvant.

The invention further provides application of the immunogenic composition in preparation of a medicament for immunizing porcine parvovirus and/or Japanese encephalitis.

Further, the content of the immunogenic composition in the medicine is 70-90 mug/mL, and one part is 2 mL.

The invention has the following beneficial effects:

the invention optimizes the nucleotide sequence of the nucleotide sequence for coding the porcine parvovirus VP2 protein and the Japanese encephalitis E protein, combines a special signal peptide GP64, effectively improves the secretion expression level of the target protein porcine parvovirus VP2 protein and the Japanese encephalitis E protein, has completely consistent structure, function and natural state of the target protein and high expression level, and furthest retains the immunogenicity of the target protein.

The invention further prepares the porcine parvovirus VP2 protein and the Japanese encephalitis E protein into a bivalent vaccine by matching with an adjuvant, and experimental verification proves that the bivalent vaccine has better immunogenicity and safety, exceeds the existing porcine parvovirus inactivated vaccine and Japanese encephalitis attenuated vaccine, and can be well applied to the immunization of the porcine parvovirus and the Japanese encephalitis.

Drawings

FIG. 1 is a diagram showing the results of enzyme digestion identification provided in example 1 of the present invention; wherein the left graph is the enzyme digestion identification result of pFastdeal-PPV VP2, and the right graph is the enzyme digestion identification result of pFastdeal-JEV E; in the two, Lane 1 is DL15000 marker, and Lane 2 is enzyme-cleaved band;

FIG. 2 is a result of detecting a purified target protein according to example 1 of the present invention; wherein the left panel is PPV VP2, lane 1 is sf9 cell control, lane 2 is GP64-VP2, lane 3 is VP2, and the right panel is JEV E;

FIG. 3 shows the result of detecting the PPV VP2 HI antibody and the JEV E neutralizing antibody after the porcine parvovirus VP2 and the Japanese encephalitis virus E protein bigeminy subunit vaccine is inoculated on a mouse, which is provided by the embodiment 2 of the invention;

FIG. 4 shows the comparison result of the porcine parvovirus VP2 and Japanese encephalitis virus E protein bivalent subunit vaccine with the PPV VP2 HI antibody level of the PPV inactivated vaccine and the JEV E neutralizing antibody level of the JEV E attenuated vaccine after the inoculation of pigs, which are provided by the embodiment 2 of the invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

(I) construction of recombinant transfer vectors pFastBac-PPV VP2 and pFastBac-JEV E

1. Obtaining the target genes PPV-VP2, JEV-E

According to the insect cell High Five^TMThe preference of the protein is that 1836bp PPV-VP2 and 1467bp JEV-E are artificially synthesized, an AcMNPV GP64 signal peptide sequence is introduced at the amino terminal, and an 8 His tag peptide sequence is introduced at the carboxyl terminal, so that the protein lays a foundation for the affinity purification of target proteins. The synthetic gene sequences are shown in SEQ ID NO 1 and SEQ ID NO 2. The synthesized objective genes PPV-VP2 and JEV-E were placed on plasmid vectors pUC-18 (Invitrogen), which were obtained as pUC-PPV VP2 and pUC-JEV E.

2. Construction of recombinant transfer vectors pFastBac-PPV VP2 and pFastBac-JEV E

Adopting a conventional recombinant plasmid construction process and method to construct recombinant transfer vectors pFastBac-PPV VP2 and pFastBac-JEV E:

enzyme digestion: this example used BamHI, EcoRI two enzymes (from TAKARA) to cut sites, and artificially synthesized genes PPV VP2 and JEV E were subjected to double digestion from pUC-PPV VP2 and pUC-JEV E; the transferred vector pFastdial was digested with the same endonuclease at the same time. The system is as follows:

TABLE 1 digestion System for transfer vector pFastDual

After the digestion, the fragments were separated by agarose gel electrophoresis and recovered.

Connecting:

TABLE 2 connection System

③ conversion: the ligation products were transformed into E.coli competent DH10B and plated.

Extracting recombinant plasmids pFastdeal-PPV VP2 and pFastdeal-JEV E: selecting monoclonal colony from the plate, extracting plasmid with plasmid extracting kit, and enzyme digestion identification with BamHI and EcoRI, with the enzyme digestion identification result shown in figure 1.

(II) construction of recombinant baculovirus Ac-PPV VP2 and Ac-JEV E

1. Obtaining a recombinant shuttle vector:

respectively transferring 4 mu L of recombinant transfer plasmids pFastdial-PPV VP2 and pFastdial-JEV E into 100 mu L of escherichia coli competent DH10Bac (purchased from GiBCO BRL), carrying out ice bath for 30min, then carrying out heat shock at 42 ℃ for 90sec, then carrying out ice bath for 3min, adding 900 mu L of nonresistant SOC, carrying out resuscitation at 37 ℃ for 4h, coating the solution on a three-antibody (kanamycin, gentamicin and tetracycline) LB plate, carrying out culture at 37 ℃ for 24-48h, screening and purifying positive colonies through blue-white spots, extracting recombinant bacmid rBac-PPV VP2 and rBac-JEV E, and extracting the method: aseptically picking positive white colony in three-antibody LB liquid culture medium, culturing for 12-16h, collecting thallus, re-suspending with 0.3mL solution I (50mmol/L glucose, 10mmol/L EDTA, 25mmol/L Tris-Cl (pH8.0)), adding 0.3mL solution II (0.2mol/L NaOH, 1% SDS), slightly mixing, standing at room temperature for 5min, slowly adding 0.3mL solution III (3mol/L CH3COOK, pH 5.0), mixing, ice-bathing for 5-10min, centrifuging at 14000r/min for 10min, adding supernatant into 0.5mL isopropanol, mixing, ice-bathing for 5-10min, centrifuging at room temperature 14000r/min for 15min, washing precipitate with 70% ethanol, drying, dissolving in 40 μ L sterile water, and immediately using or storing at-20 ℃.

2. Obtaining a recombinant baculovirus:

transfecting the extracted recombinant shuttle vector into sf9 insect cells by using a liposome transfection method, culturing at 27 ℃, performing cytopathic effect after 48-72h, collecting cell culture supernatant to obtain recombinant baculovirus, and immediately using or storing the harvested recombinant baculovirus at-80 ℃ in a dark place. Transfection methods were performed according to the Liposome instructions (Cellfect II Reagent from Invitrogen).

(III) expression and purification of target proteins PPV VP2 and JEV E

1. Expression of the proteins of interest PPV VP2 and JEV E:

the harvested recombinant baculovirus was inoculated into suspension cultured insect cells High five (tm) (purchased from Invitrogen) at an inoculation dose of 0.1MOI, a cell density of 0.8 x 106/mL and a cell volume of 400 mL. And harvesting cell culture supernatant after 72-96h, and detecting the expression of target proteins PPV VP2 and JEV E by Western blot.

2. And (3) purification of the target protein:

the purification method adopts conventional nickel column affinity chromatography. The specific operation steps are as follows: taking High FiveTM cell culture supernatant 72-96h after inoculation, centrifuging at 10000rpm to remove cells and cell debris, and filtering with a 0.45 μm filter membrane to remove fine impurities; combining the filtered supernatant with a nickel column and passing the nickel column through the column; eluting with a washing buffer (50mM imidazole, 20mM Tris, 200mM NaCl) through the column; eluting with elution buffer (300mM imidazole, 20mM Tris, 200mM NaCl) through the column; the eluate was dialyzed overnight at 4 ℃ against a dialysis buffer (20mM Tris, 200mM NaCl) to obtain the objective protein. Western blot was performed to detect the purified protein and PPV VP2, which does not carry GP64 signal peptide, was expressed according to the above method for comparison. The results are shown in FIG. 2, where A compares the expression levels of PPV VP2 carrying signal peptide GP64 and not carrying signal peptide GP64, and B is the result of JEV E detection.

Example 2

Preparation of porcine parvovirus VP2 and encephalitis B virus E protein bigeminy genetic engineering vaccine

In this example, the PPV VP2 and JEV E proteins purified in example 1 were filtered after concentration determination, mixed uniformly in equal amounts, emulsified with ISA 15VG adjuvant (purchased from SEPPIC corporation) in a ratio of 4:1 to prepare subunit bivalent vaccines, so that the antigen content of PPV VP2 and JEV E in each milliliter of vaccine was 40ug, and stored at 4 ℃ for further use.

(II) safety evaluation of porcine parvovirus VP2 and encephalitis B virus E protein bigeminy genetic engineering vaccine

10 female Balb/C mice, 16-18g, were purchased and divided into A, B groups of 5 mice each. Injecting 0.3ml of VP2 prepared in the step (I) and E protein combined vaccine into each mouse in the group A subcutaneously; group B Each mouse was injected with 0.3ml of dialysis buffer (20mM Tris, 200mM NaCl); after 14 days of continuous observation, the two groups of mice have the same state and have no abnormal response, and the result shows that the vaccine is safe for the mice.

About 2 months old PPV and JEV negative pigs were purchased 6 pigs and randomized into A, B groups of 3 pigs each. Immunizing 1 pig in group A, injecting 2ml of VP2 prepared in step one and E protein combined vaccine into neck muscle, and immunizing for the second time after 3 weeks; group B was not immunized and served as a negative control. And continuously observing 28 days after the second immunization, wherein the health condition of the immunized pig in the observation period is good and is consistent with that of the non-immunized pig, and any local or systemic adverse reaction caused by vaccine injection does not occur, so that the result shows that the vaccine is safe for the pig of the body animal.

(III) evaluation of effectiveness of porcine parvovirus VP2 and encephalitis B virus E protein bigeminy subunit vaccine

1. Effectiveness evaluation of porcine parvovirus VP2 and encephalitis B virus E protein bigeminy subunit vaccine in mice

20 female Balb/C mice, 6-8 weeks old, were purchased and randomly divided into A, B groups of 10 mice each. Group A mice were injected subcutaneously into the back of each mouse at 0.2ml, boosted 2 weeks later, and group B was not immunized. Blood serum was collected and separated for hemagglutination titer determination and neutralizing antibody determination at 2 weeks of primary immunization, two weeks of secondary immunization, and four weeks of secondary immunization, respectively, and the PPV VP2 HI antibody level detection and the JEV E neutralizing antibody detection are shown in FIG. 3.

2. Porcine parvovirus VP2 and encephalitis B virus E protein bigeminy subunit vaccine for effectiveness evaluation in pig body

To further evaluate the immunopotency of the bivalent subunit vaccine prepared above against the bulk animal pigs, 16 pigs of PPV and JEV negative of about 2 months of age were purchased and randomized into 4 groups of 4 pigs each. Group 1 pigs are immunized with 1 part (2mL) of the porcine parvovirus VP2 and encephalitis B virus E protein bigeminy subunit vaccine, group 2 pigs are immunized with 1 part of PPV inactivated vaccine (prepared from Wuhan pre-family tennin-porcine parvovirus inactivated vaccine WH-1 strain and commercially available) and group 3 pigs are immunized with 1 part of JEV attenuated vaccine (prepared from Wuhan pre-family Yining-porcine encephalitis B live vaccine SA-14-14-2 strain and commercially available), neck intramuscular injection is carried out, and the second immunization is carried out after 3 weeks; group B was not immunized and served as a negative control. The anterior vena cava was collected 7 days, 14 days, 28 days and 42 days after the initial immunization, and HI titer test and neutralizing antibody test were performed. The detection results are shown in figure 4. The result shows that the vaccine has good immunogenicity, can stimulate an organism to generate protective neutralizing antibodies, and the level of the antibodies generated by the bivalent subunit vaccine of the porcine parvovirus VP2 and the Japanese encephalitis virus E protein is equivalent to the immune effect of the JEV attenuated single vaccine and superior to the immune effect of the PPV inactivated vaccine.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims

1. An immunogenic composition, comprising: porcine parvovirus VP2 protein and Japanese encephalitis E protein;

the porcine parvovirus VP2 protein and the Japanese encephalitis E protein are expressed by utilizing SEQ ID NO:1 and SEQ ID NO:2 is obtained by expressing in an insect-baculovirus eukaryotic expression system.

2. The immunogenic composition according to claim 1, wherein the molar ratio of the porcine parvovirus VP2 protein to the Japanese encephalitis E protein is 0.5-1.5: 1-2.

3. The immunogenic composition according to claim 2, wherein the porcine parvovirus VP2 protein is 20-40 μ g/mL, and the Japanese encephalitis E protein is 40-60 μ g/mL.

4. The immunogenic composition of any one of claims 1-3, wherein the eukaryotic expression system comprises pFastdual vectors and/or insect cells High Five vectors^TMExpression of the target protein is carried out.

5. A porcine parvovirus and japanese encephalitis bivalent subunit vaccine comprising the immunogenic composition of any of claims 1 to 4 and an adjuvant.

6. The porcine parvovirus and Japanese encephalitis bigeminy subunit vaccine according to claim 5, characterised in that the adjuvant is ISA 15VG adjuvant, and the mass ratio of the sum of the porcine parvovirus VP2 protein and Japanese encephalitis E protein to the adjuvant is 3-5: 1.

7. The method of preparing the porcine parvovirus and encephalitis b bivalent subunit vaccine of claim 5 or 6, comprising:

8. The method of claim 7, comprising:

9. Use of an immunogenic composition according to any one of claims 1 to 4 in the manufacture of a medicament for the immunization of porcine parvovirus, and/or Japanese encephalitis.

10. The use according to claim 9, wherein the immunogenic composition is present in the medicament in an amount of 70 to 90 μ g/mL.